This invention relates to a new family of receptors, growth hormone secretagogue-related receptors (GHSR-Rs), nucleic acids encoding these receptors, and to the use of these receptors to identify ligands that modulate GHSR-R function.
Growth hormone secretagogues (GHSs) and secretagogue-like compounds, both peptide and non-peptide, bind to and exert their biological effects (i.e., release of growth hormone (GH)) through a G protein-coupled receptor (GPC-R) distinct from the receptors for growth hormone releasing hormone (GHRH) and somatostatin (SST) (Pong et al., 1996 Mol. Endocrin. 10:57-61). The molecular cloning of the growth hormone secretagogue receptor (GHS-R) capitalized on the pivotal observation that GHSs transduce their signal through activation of the phospholipase C pathway (Cheng et al., 1991 Endocrinology 129:3337-3342; Howard et al., 1996 Science 273:974-977). cDNA and genomic DNA cloning from human, swine, and rat showed that the GHS-R is a remarkably conserved protein of 364/366 acids containing 7 putative alpha-helical transmembrane (TM) domains, a signature feature of GPC-R""s (Howard et al. 1996; McKee et al., 1997 Mol. Endocrin. 11:415-423). In all species evaluated, the GHS-R is encoded by a single highly-conserved gene containing one intron, placed at the C-terminal end of TM domain 5. The GHS-R exhibits the highest sequence similarity to the receptors for neurotensin (NT-R) with sequence identity of 34%. The biology of the growth hormone secretagogues (GHSs) is still in a relatively early stage of development. Research is focused on identification of the GHS natural ligand system and understanding the role of the GHS-R in brain regions (substantia nigra, dentate gyrus, hippocampus) other than those traditionally thought to be involved in GH secretion (Bennett et al. 1997; Guan et al. 1997).
The identification of other G-protein coupled receptors points to the existence of a new natural ligand pathway perhaps divergent from the neuropeptide neurotensin and the GHS natural ligand. Two new human full-length GPC-R""s entitled GPR38 and GPR39 were cloned having 52% and 32% protein sequence identity to the human GHS-R, respectively (McKee et al. 1997).
It would be desirable to identify other GPC-Rs perhaps impactful on GH release and elucidate their functions. It would also be desirable to identify ligands particular to these receptors that play an important role in these and other associated pathways.
One peptide for which receptors have not been molecularly identified to date is neuromedin U. Neuromedin U (NMU) is a neuropeptide first isolated from porcine spinal cord in two molecular forms, one containing 25 amino acids (NMU-25) and the other one 8 amino acids (NMU-8); Minamino et al., 1985 Biochem Biophs Res Commun130:1078-85. It was subsequently isolated from rat (NMU-23), human (NMU-25), frog (NMU-25), dog (NMU-8 and NMU-25), rabbit (NMU-25), and chicken (NMU-25); Domin et al., 1986 Biochem Biophys Res Commun 140:1127-34; Conlon et al., 1988 J Neurochem 51:988-91; Minamino et al., 1988 Biochem Biophy Res Commun 156:355-60; Domin et al., 1989 J Biol Chem 26420881-5; O""Harte et al., 1991 Peptides 12:11-5; Kage et al., 1991 Regul Pept 33:191-8; and Domin et al., 1991 Regul Pept 41: 1-8. Mammalian NMUs share a common C-terminal sequence-Phe-Leu-Phe-Arg-Pro-Arg-Asn-amide which appears to be essential for its biological activities. NMU is distributed both in the gastrointestinal tract and the central nervous system (CNS).
In the rat, the highest concentration of NMU was found in the ileum, followed by the pituitary, hypothalamus, spinal cord, thyroid, and the genitourinary tract. Immunohistochemistry studies showed that NMU immunoreactivity in the gut was only found in nerve fibers, mainly in the myenteric and submucous plexuses, and in the mucosa of all areas except stomach while no NMU immunoreactivity was found in endocrine cells. In the rat brain, NMU immunoreactivity was found in fibers widespread throughout the brain with the exception of the cerebellum.
Human and rat genes encoding NMU precursor have been isolated. Both encode NMU at the C-terminus and other potential peptide products in the middle; Lo et al., 1992 Mol Endocrinol 6:1538-44; Austin et al., 1995 J Mol Endocrinol 14:157-69.
High affinity NMU binding was characterized in rat uterus, and was shown to be sensitive to GTP-xcex3-S (Nandha et al., 1993 Endocrinology 133:482-6), suggesting the receptor for NMU was a G-protein coupled receptor. However, no receptor of NMU has been molecularly identified so far.
The physiological role of NMU remains largely unrecognized. It can cause potent contraction of smooth muscle, increase arterial blood pressure, modify intestinal ion transport, and at low doses stimulates the function and growth of the adrenal cortex. NMU was also shown to reduce the blood flow in superior enteric artery and portal vein while increase blood flow slightly in pancreatic tissue. Nevertheless, NMU is the only neuromedin without a receptor cloned nor a great deal of knowledge obtained concerning its pharmacology and physiology.
It would be most desirable to identify a G-protein coupled receptor responsive to neuromedin U or ligands sufficiently similar thereto. A receptor responsive to neuromedin U would greatly facilitate our understanding of the physiological mechanisms of neuromedin U and other ligands sufficiently similar thereto.
This invention relates to a new family of G protein-coupled receptors, growth hormone secretagogue-related receptors (GHSR-Rs) free from receptor-associated proteins, which exhibit moderate protein sequence identity (33 and 29%) to both the growth hormone secretagogue receptor (GHS-R) and the neurotensin receptor (NT-R) type 1, respectively. Particularly, the full-length mouse and human GHSR-Rs have been identified. These newly identified receptors are expressed in a diverse set of tissues. A further aspect of this invention is the above receptors which are isolated or purified.
Another aspect of this invention are GHSR-Rs which are encoded by substantially the same nucleic acid sequence, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native form. These variant forms may have different and/or additional functions in animal physiology or in vitro in cell based assays.
Growth hormone secretagogue related receptors (GHSR-Rs) are proteins containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify (e.g., by deletion) many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. This invention specifically includes such modified functionally equivalent GHSR-Rs as well as receptors comprising the binding domain of a GHSR-R of this invention.
Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.
Another aspect of this invention are nucleic acids which encode growth hormone secretagogue related receptors (GHSR-Rs). More specifically, the invention relates to nucleic acids comprising the sequences of SEQ ID NOs: 1 and 3 as well as those which hybridize to same under highly stringent conditions. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode a GHSR-R.
Yet another aspect of this invention relates to vectors which comprise nucleic acids encoding a GHSR-R. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode a GHSR-R. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
A further aspect of this invention are host cells which are transformed with a gene which encodes a growth hormone secretagogue related receptor. The host cell may or may not naturally express a GHSR-R on the cell membrane. Preferably, once transformed, the host cells are able to express a growth hormone secretagogue related receptor on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 or VERO cells are preferred, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used. Both cell lines transformed to express the GHSR-R receptor and those naturally expressing the receptor are included for use within the following assays.
One further aspect of this invention is a method of identifying ligands comprising contacting cells expressing the GHSR-R receptor in accordance with the instant invention with a compound suspected of being a ligand specific for said receptor and determining whether binding occurs, binding constituting a positive indication of the presence of a ligand.
Another aspect of this invention is a method of identifying ligands for GHSR-R which comprises contacting cells expressing the GHSR-R receptor with a compound suspected of being a ligand specific for said receptor in the presence of jelly fish aequorin or other suitable reporter responsive to Ca2+ mobilization, and monitoring for luminescence or other signal from the reporter indicating activation of the receptor, activation constituting a positive indication of the presence of a ligand.
Another aspect of the instant invention is a method of identifying ligands for GHSR-R which comprises contacting cells expressing the GHSR-R receptor with a compound suspected of being a ligand specific for said receptor, and monitoring for changes in concentration of intracellular cyclic AMP (cAMP); an increase in cAMP constituting a positive indication of the presence of a ligand.
An additional aspect of the invention is a method for determining whether a substance is a potential agonist or antagonist of GHSR-R comprising contacting cells expressing the GHSR-R receptor with labeled neuromedin U in the presence and in the absence of the substance, and measuring the binding of neuromedin U to GHSR-R, where if the amount of binding of neuromedin U is more or less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of GHSR-R, respectively.
A further aspect of the instant invention is a method of determining whether a substance is a potential agonist of GHSR-R which comprises contacting cells expressing the GHSR-R receptor with the substance in the presence of jelly fish aequorin or other suitable reporter responsive to Ca2+, and monitoring for luminescence or other signal from the reporter indicating activation of the receptor; activation constituting a positive indication of the presence of an agonist.
Another aspect of the instant invention is a method of determining whether a substance is a potential antagonist of GHSR-R which comprises contacting cells expressing the GHSR-R receptor first with the substance and then with neuromedin U in the presence of jelly fish aequorin or other suitable reporter responsive to Ca2+, and monitoring for luminescence or other signal from the reporter indicating activation of the receptor; where if the amount of luminescence or signal is less in the presence of the substance than in the absence of the substance, then the substance is a potential antagonist of GHSR-R.
Another aspect of the instant invention is a method of determining whether a substance is a potential agonist of GHSR-R which comprises contacting cells expressing the GHSR-R receptor with the substance, and monitoring for changes in cyclic AMP (cAMP); an increase in cAMP constituting a positive indication of an agonist.
Another aspect of the instant invention is a method of determining whether a substance is a potential antagonist of GHSR-R which comprises contacting cells expressing the GHSR-R receptor with the substance, and monitoring for changes in cyclic AMP (cAMP); a marginal to no increase in cAMP constituting a positive indication of an antagonist.
Another aspect of the instant invention is a method for the treatment or prevention of obesity which comprises administering to a mammal in need of such treatment or prevention an effective amount of neuromedin U or a GHSR-R agonist.
A further aspect of the instant invention is a method of decreasing food intake of a mammal which comprises administering to said mammal an effective amount of neuromedin U or a GHSR-R agonist.
Yet one further aspect of this invention is a method of determining whether a compound binds to both growth hormone secretagogue receptor (GHS-R) and growth hormone secretagogue related receptor (GHSR-R) which comprises contacting both GHS-R and GHSR-R with the compound or ligand of interest and determining whether binding occurs to both receptors.